高糖抑制蛋白KIAA0753和CCSAP表达并通过钙信号阻断小鼠胚胎成骨细胞前体细胞MC3T3-E1的成骨分化  

High glucose inhibits expression of KIAA0753 and CCSAP proteins anddisrupts osteoblast differentiation in mouse embryonic osteoblast progenitors MC3T3-E1 through impaired calcium signal transduction

作　　者：王吉春 钱正霞 李梦雪 汪长东[1,2] WANG Ji-chun;QIAN Zheng-xia;LI Meng-xue;WANG Chang-dong(Molecular Medicine and Cancer Research Center,2.Dept of Biochemistry and Molecular Biology,College of Basic Medical Sciences,3.Dept of Anesthesiology,the First Affiliated Hospital of Chongqing Medical University,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学基础医学院分子医学与肿瘤研究中心,重庆400016 [2]重庆医科大学生物化学与分子生物学教研室,重庆400016 [3]重庆医科大学附属第一医院麻醉科,重庆400016

出　　处：《中国药理学通报》2025年第3期456-465,共10页Chinese Pharmacological Bulletin

基　　金：2022年度教育部“春晖计划”合作科研项目(No HZKY20220215);重庆市科委面上项目(No CSTB2022NSCQ-MSX0945)。

摘　　要：目的探究高糖对蛋白KIAA0753和CCSAP的影响,以及高糖条件下KIAA0753和CCSAP与成骨分化以及钙信号通路三者之间的联系。方法利用5.5和25 mmol·L^(-1)葡萄糖浓度的成骨培养基诱导小鼠胚胎成骨细胞前体细胞(MC3T3-E1),通过Western blot检测KIAA0753和CCSAP蛋白表达情况;转染过表达质粒进入人胚肾细胞(HEK-293T),免疫共沉淀检测蛋白KIAA0753和CCSAP相互作用;利用成骨培养基分别在不同葡萄糖浓度和不同诱导时间处理MC3T3-E1细胞,试剂盒检测碱性磷酸酶(alkaline phosphatase,ALP)活性,Western blot检测KIAA0753、CCSAP、骨桥蛋白(osteopontin,OPN)、骨钙素(osteocalcin,OCN)等蛋白的表达情况,然后再设置5.5、25 mmol·L^(-1)和25 mmol·L^(-1)+OE-CCSAP组MC3T3-E1细胞,依次检测KIAA0753、OCN、OPN蛋白的表达情况以及ALP活性;利用高通量基因表达数据库(gene expression omnibus,GEO)中糖尿病鼠模型数据集筛选差异基因进行生物信息学分析;对MC3T3-E1细胞分别设置5.5、25 mmol·L^(-1)和25 mmol·L^(-1)+OE-KIAA0753组,Western blot检测钙离子-钙调蛋白依赖性蛋白激酶Ⅱ(calcium/calmodulin-dependent protein kinaseⅡbeta,CAMK2B)、受磷蛋白(phospholamban,PLN)蛋白的表达。结果与5.5 mmol·L^(-1)组相比,25 mmol·L^(-1)抑制成骨细胞中KIAA0753和CCSAP蛋白表达,且蛋白KIAA0753和CCSAP存在相互作用。同时25 mmol·L^(-1)也抑制成骨细胞ALP、OPN、OCN蛋白表达,在25 mmol·L^(-1)的条件下过表达CCSAP上调KIAA0753、OCN、OPN、ALP的表达。糖尿病鼠模型的差异基因主要富集在“信号受体、信号调控”等方面。25 mmol·L^(-1)葡萄糖抑制成骨细胞中CAMK2B、PLN蛋白的表达,且在25 mmol·L^(-1)的条件下过表达KIAA0753上调CAMK2B、PLN蛋白的表达。结论高糖抑制KIAA0753和CCSAP蛋白表达,抑制小鼠胚胎成骨细胞前体细胞成骨分化和钙信号传导,过表达CCSAP挽救高糖对成骨分化的抑制作用以及过表达KIAA0753逆转高糖对钙信号通路Aim To explore the effects of high glucose on KIAA0753 and CCSAP and the relationship between KIAA0753 and CCSAP and osteogenic differentiation and calcium signaling pathway under high glucose conditions.Methods Mouse embryonic osteoblast precursor cells(MC3T3-E1)were induced by osteoblast medium with glucose concentrations of 5.5 and 25 mmol·L^(-1),and the protein expressions of KIAA0753 and CCSAP were detected by Western blot.The overexpressed plasmid was transfected into human embryonic kidney cells(HEK-293T),and the interaction between KIAA0753 and CCSAP was detected by co-immunoprecipitation.MC3T3-E1 cells were treated in the osteogenic medium with different glucose concentrations and induction times.Alkaline phosphatase(ALP)activity was detected with a kit.The expression of KIAA0753,CCSAP,osteopontin,osteocalcin,and other proteins were assessed using Western blot.and then 5.5,25 mmol·L^(-1),and 25 mmol·L^(-1)+OE-CCSAP three groups of MC3T3-E1 cells were set.The expression of KIAA0753,OCN,OPN protein,and ALP activity were detected successively.The diabetic mouse model dataset in Gene Expression Omnibus was used to screen differential genes for bioinformatics analysis.MC3T3-E1 cells were set up in three groups,5.5,25 mmol·L^(-1),and 25 mmol·L^(-1)+OE-KIAA0753,respectively.The calcium/calmodulin-dependent protein kinaseⅡbeta(CAMK2B)and phospholamban(PLN)were detected by Western blot.Results Compared with the 5.5 mmol·L^(-1) group,25 mmol·L^(-1) inhibited the expression of KIAA0753 and CCSAP proteins in osteoblasts,and there was an interaction between KIAA0753 and CCSAP.At the same time,25 mmol·L^(-1) also inhibited the expression of ALP,OPN,and OCN proteins in osteoblasts.Overexpression of CCSAP at 25 mmol·L^(-1) up-regulated the expression of KIAA0753,OCN,OPN,and ALP.The differential genes of the diabetic mouse model were mainly concentrated in the aspects of“signal receptor and signal regulation”.25 mmol·L^(-1) glucose inhibited the expression of CAMK2B and PLN proteins in osteoblasts,and ove

关 键 词：糖尿病 KIAA0753 CCSAP 蛋白相互作用 成骨分化 钙信号 

分 类 号：R-332[医药卫生] R348.1R329.24R394.2R587.1R977.6